This invention relates to improvements in animal cell culture, particularly to improvements in methods for growing animal cells and nutrient media therefor.
The use of animal cell culture for the mass production of cell products such as immunoglobulins, hormones and enzymes is becoming increasingly important from a commercial point of view, and currently there is considerable effort devoted to the development of cell culture techniques for the optimisation of the large scale production of these materials.
Animal cells in culture require a basal nutrient mixture of salts, sugars, amino acids and vitamins. Usually the mixture is supplemented with a biological fluid or extract, in the absence of which most cells lose viability or fail to proliferate. The most commonly used supplement is serum.
The use of supplements, however, is not very satisfactory, since their generally undefined nature, and the variations that can exist between batches of a given type, can affect the success and reproducibility of a culture. There have thus been numerous attempts to identify the active factors in supplements such as serum, with a view to providing a better defined medium to support the growth of cells in culture. To date, this approach has met with limited success, largely due to the complex nature of biological supplements and the very small amounts of active factors that they contain.
A number of supplement-free media have been described, however, some of which are available commercially [see for example Murakami et al, Proc. Natl. Acad. Sci. USA 79, 1158-1162 (1982); Darfler et al., Exp. Cell Res. 138, 287-295 (1982) and International Patent Specification No. WO 90/03430].
Supplement-free media generally contain a complex mixture of amino acids, salts, vitamins, trace elements, carbohydrates and other growth supporting components such as albumin, insulin, glutamine, transferrin, ferritin and ethanolamine [see for example U.S. Pat. No. 4,816,401]. When cultured in such media, animal cells remain viable for a finite period of time, until one or more essential nutrients in the medium become exhausted. At such time the medium may be supplemented with a feed containing one or more energy sources and one or more amino acids [see for example International Patent Specification No. WO 87/00195]. In this way the culture may be prolonged to increase yield of cells or cell products.
Metal ions, especially ferrous and ferric ions, are essential for animal cell metabolism, and are present in culture media as components of undefined supplements such as serum, or as components of salts and trace elements included in supplement-free media. Cellular demand for metal ions can become high in animal cell culture, especially when high cell densities are reached and in practice this means that metal ions need to be made continuously available in culture to support the growth and viability of cells. To achieve this in a supplement-free medium high concentrations of a simple salt of the metal can be used, but it is often necessary for the metal to be in a chelated form in the medium to facilitate cellular uptake of the metal and/or to avoid the solubility and toxicity problems which can be associated with high metal ion concentrations.
To supply sufficient iron to cells growing in supplement-free media, simple or complex iron salts such as ferrous sulphate, ferric chloride, ferric nitrate or ferric ammonium citrate have been used, where necessary often in combination with a chelating agent. Particular iron chelating agents which have been used in cell culture include the natural proteins transferrin and ferritin; organic acids such as citric acid, iminodiacetic acid and gluconic acid; pyridoxal isonicotinoyl hydrazone; and aurin tricarboxylic acid.
A number of factors are important in selecting an iron chelating agent for general use in supplement-free media for animal cell culture. Thus, the chelating agent must have an appropriate binding affinity for the iron and be able to transport it efficiently across the cell membrane. It must also be cheap, readily available and non-toxic. Increasingly importantly, the chelating agent should be of synthetic, not animal, origin to avoid any possible unwanted contamination of any desired cell product and a consequent increase in the cost of recovery of a pure product. None of the above-mentioned chelating agents meets all of these criteria.
We have now found that 2-hydroxy-2,4,6-cycloheptatrien-1-one meets all of these criteria and may be used advantageously in animal cell culture to support the growth of cells. In particular, we have found that its use can support growth in agitated cell culture, where it is necessary to use low iron concentration to avoid toxicity problems, and where the use of other recognised chelating agents such as citrate and gluconate has failed. We have used this discovery to develop a medium and a process for the growth of animal cells.
Thus, according to one aspect of the invention, we provide a nutrient animal cell culture medium comprising assimilable sources of carbon, nitrogen, amino acids, iron and other inorganic ions, trace elements and optionally lipids and growth promoters or regulators in admixture with 2-hydroxy-2,4,6-cycloheptatrien-1-one or a derivative thereof.
In general the nutrient medium may be any known basal medium or variants thereof which will support the continuous growth of animal cells and/or sustain them during a stationary phase, to which 2-hydroxy-2,4,6-cycloheptatrien-1-one [hereinafter sometimes referred to as tropolone] or a derivative thereof has been added. Known basal media and variants thereof include for example Dulbecco""s Modification of Eagle""s Medium (DMEM), Iscove Modified Dulbecco""s Medium, Ham""s Medium, Roswell Park Memorial Institute Medium (RPMI) and Fischer""s Medium, or those described by Hu et al. in Biotechnol. Bioeng. (1985), 27, 585-595; by Crespi and Thilly in Biotechnol. Bioeng. (1981), 23, 983-993, and by Van Wezel in Dev. Biol. Stand. (1977), 37, 143-147. In one preferred aspect, the medium is a protein-free medium.
The tropolone or derivative thereof is generally present in the medium according to the invention at a concentration sufficient to support the growth and viability of the cells. The exact concentration may vary depending on the cell line in use and the other media components present, but may be easily determined using preliminary small scale tests in accordance with conventional practice. Thus, for example, for any chosen medium cells may be cultured on a small scale in the presence of a range of tropolone concentrations and the optimum concentration determined by observing the effect of different concentrations on cell growth and viability.
In general, the tropolone or tropolone derivative will be present in an excess molar concentration to the iron present in the medium for example at a molar ratio of around 5 to 1 to around 70 to 1, for example around 10 to 1 to around 70 to 1. Thus for example where the iron concentration in the medium is around 0.3 xcexcM, the tropolone or derivative thereof may be employed at a concentration of around 1.5 xcexcM to around 20 xcexcM, e.g. around 3 xcexcM to around 20 xcexcM. The iron may be present as ferrous or ferric ions, for example resulting from the use of simple or complex iron salts in the medium such as ferrous sulphate, ferric chloride, ferric nitrate or in particular ferric ammonium citrate.
Tropolone derivatives for use in the media according to the invention in general are those derivatives which are capable of chelating ferrous or ferric ions. Particular derivatives include those wherein one or more ring carbon atoms of tropolone are substituted by aliphatic, aromatic or heteroaromatic groups, e.g. by alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl or heteroaralkynyl groups. Tropolone or derivatives thereof are either commercially available [e.g. from the Aldrich Chemical Co.] or may be prepared using known literature procedures.
The media according to the invention may be prepared by appropriate mixture of individual components using conventional procedures and may either be provided in liquid form, or in dry form for reconstitution before use with an appropriate buffer, e.g. a bicarbonate buffer. In preparing the media according to the invention, it is advisable to avoid the use of a concentrated liquid mixture of tropolone and iron.
The media according to the invention may be used to culture animal cells. Thus according to a further aspect of the invention we provide a nutrient animal cell culture medium comprising assimilable sources of carbon, nitrogen, amino acids, iron and other inorganic ions, trace elements and optionally lipids and growth promoters or regulators in admixture with 2-hydroxy-2,4,6-cycloheptatrien-1-one or a derivative thereof for the continuous growth of animal cells.
The media according to the invention are particularly suitable for the continuous growth of animal cells in an agitated culture, particularly at a low iron concentration, e.g. at an iron concentration of around 0.3 xcexcM.
The animal cells which may be cultured according to the invention may be for example genetically engineered cells, lymphoid cells e.g. myeloma cells, or hybridoma or other fused cells. Particular cell types include cells of human, rat, mouse or hamster origin. The medium according to the invention is particularly suitable for use with lymphoid cells, especially myeloma cells, particularly of mouse origin, especially NS/O cells.
The media according to the invention may be used to culture animal cells to obtain an animal cell product. Thus according to a further aspect of the invention, we provide a process for obtaining an animal cell product by cell culture which comprises the steps of (1) culturing animal cells which produce said product in a nutrient culture medium comprising assimilable sources of carbon, nitrogen, amino acids, iron and other inorganic ions, trace elements and optionally lipids and growth promoters or regulators in admixture with 2-hydroxy-2,4,6-cycloheptatrien-1-one or a derivative thereof, (2) continuing the culture until said product accumulates and (3) recovering said product.
Cell products which may be obtained according to the invention include any products which are produced by cultured animal cells. Typical products include polypeptides and proteins, for example immunoglobulins such as monoclonal and recombinant antibodies and fragments thereof, hormones such as erythropoietin and growth hormone, e.g. human growth hormone, lymphokines such as interferon, interleukins such as interleukin 2, 4, 5 and 6 and industrially and therapeutically useful enzymes such as tissue plasminogen activator.
In the process according to the invention, the animal cells may generally be cultured in suspension in the culture medium in a suitable culture vessel, for example a stirred tank or airlift fermenter, using known culture techniques.
Thus, for example, a seed culture of suitable cells, obtained by conventional techniques, may be used to inoculate the culture medium. In general, the number of cells used for inoculation will be in the range 1xc3x97105 to 5xc3x97105 cells mlxe2x88x921 or less. The cells are then cultured until a desired cell density is reached and/or until sufficient product has accumulated.
The production of the desired products during the culture may be monitored using any appropriate assay for the particular product in question. Thus, for example, where the product is a polypeptide or protein, the production of this may be monitored by general assay techniques such as enzyme-linked immunoabsorbent assay or immunoradiometric assay adapted for use with the particular polypeptide or protein.
Where in the process according to the invention it is desired to isolate the cell product obtained, this may be achieved using conventional separation and purification techniques. Thus, for example, where the product is secreted by the cells into the medium it may be separated from the cells using techniques such as centrifugation and filtration and then further purified using, for example, affinity purification techniques, such as affinity chromatography. Where the product is not secreted by the cells, the above methods may still be used, but after the cells have first been lysed to release the product.